Method of reducing tissue damage at an inflammatory site using a monoclonal antibody

ABSTRACT

A method of reducing tissue injury in humans or other animal species using a monoclonal antibody to inhibit specific phagocyte functions. The monoclonal antibody is selected to bind to phagocytic leukocytes for the purpose of inhibiting migration to an inflammatory site in the body and to inhibit the adhesion and spreading of activated leukocytes reaching such an area and then, block release of toxic substances by these cells. The monoclonal antibody is administered in vivo prior or early in the course of an experience leading to an injurious inflammatory response such as can result from restoration of myocardial blood flow interrupted by an acute coronary thrombosis.

This invention relates to a novel method of reducing tissue damagemediated by inflammatory leukocyte activation by administering in vivo amonoclonal antibody specific for leukocyte adhesion-promoting molecules.More particularly, substantial reduction in the inflammatory response ofleukocytes to inflammatory signals resulting in damage to tissue orother parts of a human body is achieved by administering in vivo aselect monoclonal antibody that binds to a surface determinant expressedby granulocytes and mononuclear phagocytes of human or animal originwhereby to specifically inhibit certain adhesion-dependent leukocytefunctions which contribute to tissue injury especially, but not limitedto, in a myocardial infarct experience

BACKGROUND OF THE INVENTION

Peripheral blood in the circulatory system of a human or animal iscomprised principally of red blood cells, i.e. erythrocytes, and whiteblood cells, i.e. leukocytes. The variety of functions of leukocytes andtheir clinical relevance has generated great interest in the scientificcommunity. The family of white blood cells is comprised of neutrophils,monocytes, eosinophils, basophils and lymphocytes. Lymphocytes are ofT-lymphocyte and B-lymphocyte types which have numerous subsets.Neutrophils, eosinophils and basophils are known as "granulocytes"because of their content of cytoplasmic granules.

Neutrophils, monocytes, eosinophils and basophils are known asphagocytes because their primary function in the human immune system isto phagocytize or ingest bacteria, microorganisms and other types offoreign materials. These cells are produced from common progenitor cellsin the bone marrow of a human or animal and are known to circulate inperipheral blood and finally, enter tissues as necessary for control ofinfection or to participate in any type of inflammatory reaction.However, each of these phagocytes has different functions and behaves asa related but separate system.

The neutrophil is the most common leukocyte in human and animalperipheral blood. One microliter of normal human whole blood includes,on average, 5×10³ leukocytes of which 3,075 are neutrophils, 150 areeosinophils, 25 are basophils, 250 are monocytes, and 1,500 arelymphocytes.

In the response of granulocytes or mononuclear phagocytes to any type ofinfection or inflammation, these cells are activated first to migrate tothe appropriate area in response to chemoattractant factors, such as,certain bacterial products, complement component, and other factors.This attraction process is termed "chemotaxis". Once in an area ofinflammation or infection, granulocytes and mononuclear phagocytes mustestablish a firm attachment to their targets. For this purpose, thesecells possess a number of specific cell surface receptor glycoproteinsthat promote this interaction, such as complement, Fc, and fibronectinreceptors.

A very important family of cell surface receptor glycoproteins is theleukocyte cell adhesion molecular (LEU-CAM) family (CD11/CD18). Thisfamily is comprised of at least three (3) cell surface proteins whichhave two (2) subunits each. They share a common beta subunit of 94,000dalton molecular weight (CD18), and have different alpha subunits. Theknown members of this family are termed LFA-1 (CD 11a/CD18), Mol (CD11b/CD18), and P150,94 (CD 11c/CD18) which evidence alpha subunits of180,000, 155,000 and 150,000 dalton molecular weight, respectively. Eachof these cell surface proteins has been specifically identified throughthe use of monoclonal antibodies. The biological importance of thisfamily of surface glycoproteins has been recognized through theidentification of a human disease in which leukocytes are geneticallydeficient in this family of antigens. (Arnaout, M.A., Dana, N., Pitt,J., and Todd, R.F. III., Deficiency of two human leukocyte surfacemembrane glycoproteins (Mol and LFA-1). Fed. Proc. 44: 2664-2670 (1985).The disease is characterized by recurrent severe bacterial infectionsand deficiencies in adhesion-dependent functions such as phagocytosis,neutrophil spreading on plastic, leukoaggregation, and chemotaxis.

The Mol glycoprotein has been of particular interest as it has beenshown that this particular structure has the capacity to bind acomponent of complement termed iC3b, a fragment of the third componentof complement. Arnaout, M.A., Todd, R.F. III, Dana, N., Melamed, J.,Schlossman, S.F., and Colten, H.R., Inhibition of phagocytosis ofcomplement C3 or IgG-coated particles and of iC3b binding by monoclonalantibodies to a monocyte-granulocyte membrane glycoprotein (Mol), J.Clin, Invest. 72:171-179 (1983). Also, the Mol glycoprotein iscritically important in all of the adhesion-dependent phagocytefunctions. Different monoclonal antibodies have been shown to inhibitthe functions of the Mol glycoprotein.

Mol is a cell surface glycoprotein present on granulocytes, mononuclearphagocytes and null cells. Todd, R.F.III, Nadler, L.M. and Schlossman,S.F., Antigens on Human Monocytes, Journal of Immunology, 126: 1435-1442(1981). In humans, this molecule consists of two non-covalently linkedproteins of 155,000 and 94,000 daltons. Todd, R.F. III, van Agthoven,A., Schlossman, S.F., and Terhorst, C. Structural analysis ofdifferentiation antigens. Mol and Mo2 on human monocytes, Hybridoma1:329-337 (1982). This complex has been shown to mediate cell adhesionto a variety of surfaces including other granulocytes, endothelium, andinert substrates. Genetic deficiencies in these molecules result inrecurrent bacterial infections due to the inability of granulocytes tomediate an anti-microbial inflammatory response. Patients who aredeficient in these molecules are characterized by an elevated leukocytecount (called "leukocytosis" and functional defects in phagocyteactivity as measured in vitro by reduced or absent aggregation adhesionto substrates, chemotaxis, and phagocytosis of opsonized particles.Activation of granulocytes and monocytes by soluble inflammatorymediators increases expression of these molecules. Todd, R.F. III,Arnaout, M.A., Rosin, R.E., Crowley, C.A., Peters, W.A. and Babior, B.M.The subcellular localization of Mol (Molα; formerly gp¹¹⁰) a surfaceglycoprotein associated with neutrophil adhesion. J. Clin. Invest.74:1280-1290 (1984); Arnaout, M.A., Hakim, R.M., Todd, R.F., Dana, N.and Colten, H.R., Increased expression of an adhesion-promoting surfaceglycoprotein in the granulocytopenia of hemodialysis, New Engl. J. Med.,312: 457-462 (1985). Monoclonal antibodies directed against the Molglycoprotein effectively prevent neutrophil aggregation in vitro as wellas prevent phagocytosis. In a rat lung model of neutrophil-mediated lunginjury (acute respiratory distress syndrome [ARDS]), anti-Mol monoclonalantibody significantly inhibited plumonary endothelial damage producedby activated human neutrophils. Ismail, G., Morganroth, H.L., Todd, R.F.III, and Boxer, L.A. Prevention of pulmonary injury in isolated perfusedrat lungs by activated human neutrophils preincubated with anti-Molmonoclonal antibody, Blood 69:1167-1174, (1987).

While the inflammatory response of leukocytes is vital to the eradictionof invading microorganisms, a substantial and convincing body ofevidence indicates that inflammatory phagocytes cause damage to variousorgans and tissues when these cells are activated in vivo by solubleinflammatory factors that are generated by inciting pathological events.Harlan, J. M., Leukocyte-Endothelial Interactions, Blood 65: 513-525(1985). The adhesion and spreading of activated neutrophils andmononuclear phagocytes to vascular endothelial cells with the subsequentrelease of toxic oxidative metabolites and proteases has been implicatedin the organ damage observed in diseases, such as, adult respiratorydistress syndrome (ARDS; shock lung syndrome), glomerulonephritis, andinflammatory injury occurring after reperfusion of ischemic tissue suchas to the heart, bowel, and central nervous system. Reviewed in Harlan,J. M., ibid.) That the heart muscle or myocardium is vulnerable to theinflammatory response of activated leukocytes has been demonstrated bythe outcome of several investigations. These studies have demonstratedthat if dogs are depleted of circulating granulocytes with a neutrophilspecific antiserum [Romson, J. L., et al., Reduction of the extent ofischemic myocardial injury by neutrophil depletion in the dog,Circulation, 67: 1016-1023 (1983)] or nitrogen mustard [Mullane, K. M.et al., Role of leukocytes in acute myocardial infarction inanesthetized dogs: Relationship to myocardial salvage byanti-inflamatory drugs, J. Pharmacal. Exp. Ther. 228: 510-522 (1984)]prior to the induction of regional myocardial ischemia and reperfusion,the size of myocardial infarct that results is significantly smallercompared to dogs with normal circulating neutrophil counts. There are anumber of other studies that have shown that agents that inhibitneutrohpil activation also result in reduced myocardial infarct size.Romson, J. L. et al., The effect of ibuprofen on accumulation of111-indium labelled platelets and leukocytes in experimental myocardialinfarction, Circulation 66: 1002-1011 (1982); Bednar, M. et al.,Nafazatrom-induced salvage of ischemic myocardium in anesthetized dogsis mediated through inhibition of neutrophil function, Circ. Res. 57 :131-141 (1985).

One monoclonal antibody which evidences the capability of inhibitingadhesion-dependent functions but does not affect binding of iC3b isknown as MY904. Dana, N., Styrt, B., Griffin, J. D., Todd, R. F. III,Klempner, M. S., and Arnaout, M. A., Two functional domains in thephagocyte membrane glycoprotein Mol identified with monoclonalantibodies, J. Immunol. 137:3259-3263 (1986). Thus, the binding of themonoclonal antibody MY904 to neutrophils could specifically inhibitmigration of neutrophils to an area of inflammation or infection.Further, such specific binding of MY904 antibody could inhibit theadhesion and spreading of activated neutrophils reaching such an areaand then block the deleterious effects of toxic substances released bythe granulocyte.

The method embodying the invention utilizes the specific advantages ofthe MY904 monoclonal antibody for reducing injury in vivo. The MY904monoclonal antibody is administered in vivo in the setting of an acuteinflammatory response mediated by inflammatory leukocytes, for example,in an acute coronary thrombosis experience just prior to the restorationof myocardial blood flow to ischemic myocardium. This infusion of MY904antibody to impact on the phagocyte population in peripheral blood ortissue may inhibit or diminish the ability of these inflammatory cellsto mirgrate to the inflammatory site within the affected tissue; andfurther, may inhibit adhesion of neutrophils, for instance, in such areaso as to inhibit or minimize the potential deleterious effects of toxicsubstances released by adherent cells. For example, this procedure wasdetermined to materially reduce tissue damage in the area of myocardialinfarction after myocardial blood flow is returned.

SUMMARY OF THE INVENTION

The method of materially reducing tissue injury mediated by inflammatoryphagocytic leukocytes, such as, but not limited to, myocardial infarctsize. The monoclonal antibody MY904 is administered in vivo inanticipation of or early in the course of a potentially injuriousinflammatory response mediated by activated phagocytic leukocytes, suchas in a myocardial infarct experience, prior to the restoration ofmyocardial blood flow (interrupted due to an acute coronary thrombosis)by the action of a thrombolytic agent or surgery. The MY904 monoclonalantibody serves to inhibit certain functions of granulocytes andmononuclear phagocytes which ordinarily induce damage of tissue at aninflammatory site, such as in the area of myocardial ischemia withattendant tissue damage after reperfusion. The use of the MY904monoclonal antibody in the setting of reperfusion myocardial injury isshown to be effective in decreasing the size of anticipated infarctionby a significant percentage when administered in vivo prior toreperfusion of ischemic mycocardium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphic illustration of mean arterial blood pressure (MAP)data accumulated in practicing the method embodying the invention;

FIG. 1B is a graphic illustration of the heart rate (HR) dataaccumulated in practicing the said method;

FIG. 1C is a graphic illustration of the left circumflex blood flow(LCX) data accumulated in practicing said method;

FIG. 1D is a graphic illustration of the rate pressure product (RPP)data accumulated in practicing said method;

FIG. 2 is a graphic illustration of data accumulated to show effect ofthe invention on resulting myocardial infarct size;

FIG. 3 is a graphic illustration of data accumulated to express infarctsize as a function of the extent of the ST segment of anelectrocardiogram; and

FIG. 4 is a graphic illustration of data accumulated to show circulatingneutrophil counts during the myocardial infarction process.

PREFERRED EMBODIMENT OF THE INVENTION

The monoclonal antibody employed in the method of the invention isidentified by the designation MY904. It was developed from the fusion ofmurine spleen cells immunized with human chronic granulocytic leukemia(CGL) cells by standard procedure described by Kohler and Millstein,Nature 256: 495-497 (1975). The granulocytic leukemia cells used in theimmunization procedure were obtained from newly diagnosed patients withCGL as part of diagnostic evaluation. Blood was obtained by venipunctureand mononuclear cells separated from granulocytes and red blood cells byFicoll-Hypaque density gradient sedimentation, 1.077 g/cc. Themononuclear cell fraction was composed of immature granulocytes andblast cells. These cells were cryopreserved, and mice immunized atweekly intervals for 4 weeks with 10×10⁶ thawed mononuclear cellsinjected intraperitoneally. Three days prior to fusion, 10×10⁶ similarlytreated CGL mononuclear cells were injected intravenously into the tailvein of the mouse. For the fusion, the spleen was removed and a singlecell suspension made of splenocytes. The spleen cells were then mixedwith the NS-1 plasmacytoma cell line at a ratio of 8 spleen cells l to 1NS-1 cell in serum free media. The cells were centrifuged to a pellet,suspended in 0.5 ml. of 30% polyethylene glycol for 8 minutes at 25° C.,followed by washing of the cells one time in serum free media anddilution in HAT media prior to distribution of the cells in microtiterplates.

Monoclonal antibody producing hybridoma clones reactive with theimmunizing cell population were selected by immunofluorescence screening14 days after the infusion. Monoclonal antibody MY904 was identified asan antibody which reacted with CGL cells as well as with normal humangranulocytes, monocytes, and a fraction of large granular lymphocytes.The monoclonal antibody reacted with more than 90% of granulocytes of 10of 10 patients tested. The monoclonal antibody MY904 does not react withT lymphocytes or B lymphocytes. The monoclonal antibodyimmunoprecipitates a glycoprotein composed of 2 subunits, 155,000daltons and 94,000 daltons from surface labelled normal humangranulocytes. Dana, N., et al. Two functional domains in the phagocytemembrane glycoprotein Mol identified with monoclonal antibodies, J.Immunol. 137:3259-3263 (1986). The distribution of reactivity ofmonoclonal antibody MY904 and the molecular weight of the antigenidentified by this monoclonal antibody indicates that the antigen is theCD11b/CD18 ("Mol") glycoprotein. Functional studies have furtherindicated that monoclonal antibody MY904 does not inhibit iC3b bindingbut is a potent inhibitor of the adhesion-dependent processes,granulocyte spreading on plastic and chemotaxis (Dana et al., TwoFunctional Domains In The Phagocyte Membrane Glycoprotein Mol IdentifiedWith Monoclonal Antibodies, Journal of Immunology 137:3259-3263, 1986).In comparison with other anti-Mol monoclonal antibodies, antibody MY904was unique in that it inhibited only adhesion-dependent functions butnot binding of iC3b. Other antibodies tested include monoclonalantibodies 44, 903, 94, 17, OKM10, and Leu-15. Dana et al., ibid.

Thus, monoclonal antibody MY904 identifed the Mol granulo-cyte-monocytecell surface glycoprotein, and further binds specifically to an epitopeon that glycoprotein which is involved in adhesion-dependent processesof granulocyte/monocyte activities.

A sample of the hyrbid cell line capable of producing the MY904monoclonal antibody is on deposit with the American Type CultureCollection (A.T.C.C.) and is assigned A.T.C.C. No. HB 9510.

Studies in vitro have shown that human, canine and subhuman primateleukocytes have in common the Mol glycoprotein. Letvin, N.L., Todd, R.F.III, Palley, L.S., and Griffin, J.D. Conservation of myeloid surfaceantigens on primate granulocytes Blood 61:408-410 (1983). Further, astudy was reported wherein a leukocyte glycoprotein deficiency syndromein a dog was found to be similar to that recognized in a human. Giger,U. Boxer, L. A., Simpson, P. A., Lucchesi, B. R., and Todd, R. F. III.Deficiency of leukocyte surface glycoproteins Mol, LFA-1, and Leu M5 ina dog with recurrent bacterial infection. An animal model. Blood,69:1622-1630, 1987. The monoclonal antibody MY904 was shown to bind tothe alpha subunit (CD11b) of both dog and human leukocytes. Also,binding of the MY904 monoclonal antibody to normal dog neutrophils dideffectively inhibit neutrophil aggregation in vitro when stimulated withthe phorbol ester PMA (Giger et al, ibid). It is believed that themethod embodying the invention which employs in vivo administration of aspecified monoclonal antibody to a live human or animal suffering from aphagocyte mediated inflammatory response, such as from inducedmyocardial ischemic event with attendant reduction in myocardial damageupon subsequent reperfusion of the myocardium is unique and unexpected.

In the example of administering MY904 antibody to attenuate myocardialreperfusion injury, a group of 23 adult dogs was first prepared. Theanimals were anesthetized and their hearts were exposed through a leftthoracotomy in the fifth intercostal space and suspended in apericardial sling. The left circumflex coronary artery was instrumentedwith a calibrated electromagnetic flow probe for the continuousrecording of blood flow. Catheters were placed into the aorta for bloodpressure recording and into the left jugular vein for monoclonalantibody infusion and blood sampling. The standard limb lead 11electrocardiogram were recorded continuously.

Regional myocardial ischemia was produced by occluding the circumflexartery for ninety (90) minutes and then reperfusing the myocardium inthe presence of a critical stenosis or narrowing of a blood vessel. Thisprocedure prevents development of hemorrhagic infarction and reduces theincidence of reperfusion induced ventricular fibrillation. Afterreperfusing the myocardium for six (6) hours, the heart was electricallyfibrillated and excised. The infarct size was assessed as a percentageof the area to the myocardium at risk of infarction as well aspercentage of the total left ventricle. The ex vivo dual perfusionhistochemical staining technique described in Romson et al., Thebeneficial effects of oral ibuprotein on coronary artery thrombosis andmyocardial ischemia in the conscious dog. J. Pharm. and Exp. Therap.215:271 (1980) was employed. The cannulated circumflex was perfused with1.5% triphenyltetrazolium chloride (TPT) solution buffered with 20 mMpotassium phosphate (pH 7.4), while simultaneously perfusing theremainder of the coronary circulation with Evan's blue dye introducedinto the aorta. Both solutions were delivered to the respective vasculardistributions under a constant pressure of 100 mm mercury at atemperature of 37° C. for five minutes. The hearts were then cut into 5or 6 centimeter thick transverse sections and infarct size wasdetermined planimetrically. This accepted method of measuring infarctsize accurately demarcates viable from non-viable myocardial tissue asdetermined by histochemical reaction between TPT and myocardialdehydrogenase enzymes.

With this preparation of the animals completed, two groups of dogs werestudied. One group, 11 in number, designated the "controls" group,received 5% human serum albumin, the vehicle for the monoclonalantibody. The second group of animals, 9 in number, received thepharmaceutic grade monoclonal antibody MY904 in amounts of one (1) mg/kginfused intravenously over a ten (10) minute period (45 minutes) afterregional myocardial ischemia was induced (45 minutes prior toreinitiation of coronary blood flow). Detection of binding of the MY904antibody to the Mol antigen of dog leukocytes was monitored. Themonoclonal antibody MY904 was supplied by Coulter Immunology Division,Coulter Corporation, in Hialeah, Florida.

At 0, 85, and 120 minutes after injection of MY904 antibody into a dog,4 ml aliquots of venous blood were withdrawn from experimental subjects,placed into EDTA-containing tubes, and centrifuged for 5 minutes at 800g. Plasma was separated from the pellet and was saved for subsequentanalysis of residual monoclonal antibody. The cellular pellet wasdepleted of erythrocytes by ammonium chloride lysis, and the residualleukocytes were analyzed for the presence of pre-existing bound anti-Molantibody by immunofluorescence staining. 1-2×10⁶ leukocytes wereincubated for 30 minutes at 4° C. in buffer alone or in buffercontaining a saturating concentration of murine anti-Mol antibody. Thecells were then washed and incubated for an additional 30 minutes at 4°C. in buffer containing a saturating concentration offluoresceinconjugated goat anti-mouse immunoglobulin. Antibody binding,either as a result of in vivo administration of anti-Mol or after invitro exposure to additional anti-Mol antibody, to dog neutrophils andmonocytes was assessed by flow cytometry after selective gating on thesemyeloid cells, as determined by log forward angle versus log right anglelight scatter, using an EPICS® C flow cytometer available from CoulterCorporation of Hialeah, Florida. The fluorescence intensity of 5000cells per determination was used as a quantitative measure of monoclonalantibody binding.

To document the administration of the MY904 and anti-Mol monoclonalantibody sufficient to produce anti-Mol antibody excess in the plasma orsera of treated dogs, samples of plasma (EDTA-anticoagulated blood) orserum were analyzed. This was assessed by indirect immunofluorescenceanalysis in which 1x10⁶ Mol-positive test cells, calcium ionophoreA23187-stimulated human neutrophils, were incubated in buffer containingdog plasma or serum (1/2, 1/4, 1/8, 1/16 dilutions) for 30 minutes at 4°C. Then the cells were washed in buffer containing a saturatingconcentration of fluorescein-conjugated goat anti-mouse immunoglobulinfor an additional 30 minutes at 4° C. Monoclonal antibody binding totest cells was quantitated by flow cytometry procedures as describedherein using the EPICS® instrument.

To evaluate neutrophil accumulation in myocardial tissue, 50-200 mgsamples of myocardium were taken from the central infarct region, thenon-infarcted tissue within the area at risk, the endocardial toepicardial border zone between infarct region and area at risk and alsofrom normal non-infarcted and unstained myocardium. Tissue samples werehomogenized and assayed for myeloperoxidase content as described inBradley, P.O., et al., Measurement of cutaneous inflammation: Estimationof neutrophil content with an enzyme marker, J. Invest. Derm. 78:206-209 (1982) The myeloperoxidase content of myocardial tissue afterinfarction has been correlated with histologic evidence of neutrophilinfiltration as described in Bednar, M., et al., Circ. Res. 57: 131-141(1985); Mullane, K. M., et al., J. Pharmacololgical Methods 14:157-167(1946).

To obtain a histological assessment of infarction and neutrophilaccumulation within the myocardium, representative histological sectionsstained with hematoxylin and eosin from each heart were evaluated by anindependent qualified investigator who was unaware of the specifictreatment. The data was evaluated.

To assess effects of monoclonal antibody on isolated neutrophilaggregation, neutrophils were isolated from venous blood of untreateddogs by Ficoll-hypaque gradient techniques. Red blood cells were lysedwith buffered ammonium chloride and resuspended to a concentration of10⁷ per ml in Hank's balanced salt solution. Aggregation was assessed ina platelet aggregation profiler, Model PAP-3, BIO/DATA Corporation,Horsham, PA. Samples of neutrophils were preincubated with MY904 andnegative control antibodies and then activated with 125 ng/ml PMA.

All data were compared to respective control group in which values ofP<0.05 were considered significant.

As previously noted, twenty-three dogs were started in the study toassess the effects of anti-Mol monoclonal antibody on the myocardialinfarct size that results after regional ischemia and reperfusions. Ofthese 23 dogs, 16 were included in the final analysis of infarct sizes.Three dogs were eliminated from the study due to failure to developobjective signs of ischemia as measured by electrocardiographic changesand four dogs, 3 untreated and 1 treated with anti-Mol antibody, wereeliminated due to ventricular fibrillation.

Mean arterial blood pressure (MAP) heart rate (HR) left circumflex (LCX)blood flow and rate pressure product (RPP; MAP x HR/100) were measuredat regular intervals during the experiments to determine whether theantibody had any effects on these parameters. Data accummulated aredepicted in FIGS. 1A, 1B, 1C and 1D respectively. The two treatmentgroups had similar MAP, HR, RPP and LCX blood flow at baseline and atevery time point during the experiments. Thus, the protective effects ofthe antibody could not be attributed to changes in myocardial bloodsupply, as measured by LCX blood flow, or myocardial oxygen demand asmeasured by RPP or HR or MAP.

Infarct size was reduced by 46% by the administration of the anti-Molantibody as compared to control, as seen from the data of FIG. 2.Similarly, infarct size was significantly smaller with monoclonalantibody treatment when infarct size was expressed as a percentage ofthe total left ventricle. The percentage of the left ventricle that wasrendered ischemic, i.e., the area at risk/left ventricle, was similarbetween treatment groups. When infarct size is expressed as a functionof the extent of ST segment elevation on the electrocardiogram, thereexists a good correlation with each treatment group describing adifferent regression line as seen from the data of FIG. 3. These datasuggest that for a given severity of myocardial ischemia as measured byST segment elevation, the infarct size that results is larger in thecontrol group compared to the monoclonal antibody treated group. Thus,myocardial infarct size is reduced by monoclonal antibody treatmentindependent of the severity of ischemia.

By immunofluorescence analysis, the neutrophils of all 20 experimentalsubjects expressed the antigenic epitope detectable by the anti-MolMY904 monoclonal antibody. In the 9 dogs who received anti-Molmonoclonal antibody at each time point after antibody administration,(i.e., 40, 75, and 405 minutes after antibody administration), the serumor plasma contained residual anti-Mol antibody as assayed byimmunofluorescence staining of serum or plasma-treated Mol positive testcells. This indicated that an infusion of 1 mg/kg was sufficient toproduce antibody excess in vivo. Detectable subsaturating amounts ofbound antibody were found on the leukocytes of only 6 out of 9 dogswhich may reflect antibody dissociation from the membrane prior to assayby incubation with the fluorescein-conjugated goat anti-mouseimmunoglobulin reagent.

FIG. 4 data shows that neutrophil counts normally increase as seen inthe control group during the process of myocardial infarction. However,treatment with anti-Mol antibody suppressed this early rise incirculating neutrophil counts when the two groups are compared. Thesedata, in conjunction with the measurement of excess antibody in theblood and antibody bound to leukocytes, confirm that neutrophils fromthe antibody treated group are not being cleared from the circulation.One potential problem with the treatment of leukocytes with antibodiesis that they may be quickly cleared because of the antibody binding tothe cell surfaces. These data suggest that the treatment with thisantibody does not cause a drastic reduction in the number of circulatingneutrophils.

In an additional study, a third group of five (5) dogs was treated in anidentical manner with a negative control murine IgGl monoclonalantibody, i.e., non-reactive with dog leukocytes. We determined thatinfarct size after reperfusion was not materially different from thatexhibited by the other control group receiving HSA diluent.

The data developed from our method evidenced that prevention ofneutrophil adhesion by a monoclonal antibody, MY904, directed againstthe leukocyte adhesion promotion molecule Mol can reduce reperfusioninjury in a well characterized animal model of myocardial infarction.The data developed by the method of the invention indicate thatadministration of the anti-Mol monoclonal antibody MY904 can reducemyocardial infarct size, but not by lessening the severity of theischemia. Since the duration of ischemia was ninety (90) minutes in allinstances, the mechanism of protection by the MY904 antibody is a likelyconsequence of its effects on neutrophil adhesion with a reductionneutrophil-mediated injury of ischemic myocardium. Heretofore, it wasconsidered that the two major determinants of myocardial infarct sizewere the severity and duration of the ischemia period.

It will be appreciated from the data accumulated from practicing themethod embodying the invention that administering the monoclonalantibody MY904 to a subject within forty five minutes of regionalmyocardial ischemia materially reduces infarct size independently of theseverity of the ischemia. Further, the data shows that inhibition ofleukocyte adhesion by anti-Mol monoclonal antibody MY904 afteroccurrence of regional myocardial ischemia results in reduced myocardialreperfusion injury as measured by infarct size. The size of myocardialinfarct expressed as a percentage of the area at risk of infarction thatresulted was reduced by 46% with the MY904 monoclonal antibody treatmentcompared to the control.

For treatment of a human experiencing a coronary thrombotic incidentusing the method embodying the invention, medical experience indicatesthat a patient who experiences pain or discomfort in his chest would bebrought to a hospital usually within thirty to sixty minutes thereafteron an emergency basis. He would be examined promptly to document theoccurence of acute myocardial ischemia and the location of a coronaryartery occlusion, and treatment would be prescribed. Such treatment maybe that of infusing a thrombolytic agent (e.g., streptokinase or tissueplasminogen activator [TPA]) which might dissolve the blood clot whichcaused the acute coronary occlusion. Another treatment is surgical innature where a balloon catheter is inserted into the circulatory systemand directed to the blood clot area for eliminating the blockage. Ineiether manner of treatment, just prior to the intervention resulting inreperfusion of ischemic mycocardium (i.e., restoration of coronaryartery blood flow), a quantity of MY904 monoclonal antibody is injectedintravenously into the patient. A single intravenous dose or multipledoses may be administered to optimally attenuate the inflammatoryresponse. The MY904 monoclonal antibody thereafter functions in themanner described herein to reduce tissue damage (infarct size)independently of the severity of the ischemic event. The MY904monoclonal antibody infusion functions to reduce myocardial reperfusioninjury by inhibiting undesired neutrophil functions.

Procedural steps involved in practicing the method embodying theinvention may be varied in minor respects without departing from thescope or spirit of the invention as stated in the appended claims.

We claim:
 1. A method of treating a human or animal host with the intentof reducing tissue damage occurring at an inflammatory site in any partof the body of the host experiencing a phagocyte-mediated inflammatorycondition, said method comprising:administering in vivo a monoclonalantibody having the binding specificity of the MY904 murine monoclonalantibody produced by the cell line A.T.C.C. No. HB 9510 which will bindspecifically to an epitope expressed on the CD11b part of the CD11/CD18glycoprotein of the Mol antigen expressed on the surface of granulocytesand other phagocytic cells and will inhibit the CD11/CD18 adhesiondependent cellular interactions of such cells reflecting theirimmunological inflammatory response function which contributes to suchdamage and yet, not interfere with the binding of iC3b.
 2. The method ofclaim 1, in which said monoclonal antibody binds to an epitope on theCD11b, 155,00 dalton molecular weight peptide molecule of the Molglycoprotein expressed on the surface of such cells.
 3. The method claimof claim 1 in which said inflammatory site is located at the vascularendothelial cell interface or subcellular matrix of a body part.
 4. Themethod of claim 1 in which said inflammatory site is in endothelialtissue of a body part.
 5. The method of claim 1 in which saidinflammatory site is in a joint of the body part.
 6. The method of claim1 in which said inflammatory site is developed from a mycocardialinfarct condition.
 7. The method of claim 4 in which the monoclonalantibody is administered intravenously at a selected time period priorto or during said inflammatory condition.
 8. The method of claim 1 inwhich said monoclonal antibody binds the Mol glycoprotein ofneutrophils.
 9. A method of treating a human host with the intent ofreducing tissue damage occurring at an inflammatory site in the body ofthe human experiencing an inflammatory condition, said methodcomprising:infusing into the body prior to or during said inflammatorycondition a quantity of murine monoclonal antibody having the bindingspecificity of the MY904 monoclonal antibody developed from thehybridoma cell line A.T.C.C. No. HB 9510 which will bind specifically toan epitope expressed on the CD11b, 155,000 dalton molecular weightpeptide of the Mol antigen expressed on the surface of neutrophils andwill inhibit the CD11/CD18 adhesion dependent intercellular reactions ofneutrophils reflecting their immunological response function whichcontributes to such damage and yet, not interfere with binding of iC3b.10. A method of reducing myocardial inflammation damage in a humanpatient experiencing an acute coronary thrombosis, said patient havingbeen treated initially either medically or surgically to renewmyocardial blood flow at the inflammatory site, said method comprisingsupplying intravenously at least prior to such blood flow renewal aquantity of a murine monoclonal antibody which binds specifically anepitope expressed on CD11b part of the CD11/CD18 glycoprotein of the Molsurface antigen of neutrophils whereby to inhibit the adhesive-dependentcellular interactions of the neutrophils at said site reflecting theirimmunological response function whereby to decrease the deleteriousaction of neutrophils at said site and without interfering in thebinding of iC3b to the antibody bound target neutrophils.
 11. The methodof claim 8 in which said monoclonal antibody is the MY904 monoclonalantibody which is produced by hybridoma technology from the cell linehaving the essential characteristics of the hybrid cell line samples ondeposit with the Americal Type Culture Collection and assigned A.T.C.C.Deposit No. HB 9510.